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Published online before print April 5, 2006, 10.1110/ps.052004006
Protein Science (2006), 15:1063-1075. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study

Rahul S. Rajan1, Tiansheng Li1, Mohini Aras4, Christopher Sloey1, Weston Sutherland2, Hiromi Arai5, Robert Briddell2, Olaf Kinstler3, Alexis M.K. Lueras1, Yu Zhang1, Heather Yeghnazar1, Michael Treuheit1 and David N. Brems1

Departments of 1 Pharmaceutics,
2 Hematology,
3 Protein Science, Amgen Inc., Thousand Oaks, California 91320, USA
4 Department of Chemical Engineering, University of California, Berkeley, California 94720, USA
5 Cal State Channel Islands, Department of Biology, Camarillo, California 93012, USA

(RECEIVED November 30, 2005; FINAL REVISION February 3, 2006; ACCEPTED February 9, 2006)

Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N-terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG-GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG-GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG-GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG-GCSF and 20kPEG-GCSF. It was observed that even 5kPEG-GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG-GCSF. Biological activity measurements demonstrated that both 5kPEG-GCSF and 20kPEG-GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface.

Keywords: GCSF; protein aggregation; protein stability; polyethylene glycol; solubility


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